Nitric Oxide Participation in the Fungicidal Mechanism of Gamma Interferon-Activated Murine Macrophages againstParacoccidioides brasiliensisConidia

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Abstract
Paracoccidioidomycosis, a systemic mycosis restricted to Latin America and produced by the dimorphic fungusParacoccidioides brasiliensis, is probably acquired by inhalation of conidia produced by the mycelial form. The macrophage (Mφ) represents the major cell defense against this pathogen; when activated with gamma interferon (IFN-γ), murine Mφs kill the fungus by an oxygen-independent mechanism. Our goal was to determine the role of nitric oxide in the fungicidal effect of Mφs onP. brasiliensisconidia. The results revealed that IFN-γ-activated murine Mφs inhibited the conidium-to-yeast transformation process in a dose-dependent manner; maximal inhibition was observed in Mφs activated with 50 U/ml and incubated for 96 h at 37°C. When Mφs were activated with 150 to 200 U of cytokine per ml, the number of CFU was 70% lower than in nonactivated controls, indicating that there was a fungicidal effect. The inhibitory effect was reversed by the addition of anti-IFN-γ monoclonal antibodies. Activation by IFN-γ also enhanced Mφ nitric oxide production, as revealed by increasing NO2values (8 ± 3 μM in nonactivated Mφs versus 43 ± 13 μM in activated Mφs). The neutralization of IFN-γ also reversed nitric oxide production at basal levels (8 ± 5 μM). Additionally, we found that there was a significant inverse correlation (r= −0.8975) between NO2−concentration and transformation ofP. brasiliensisconidia. Additionally, treatment with any of the three different nitric oxide inhibitors used (arginase,NG-monomethyl-l-arginine, and aminoguanidine), reverted the inhibition of the transformation process with 40 to 70% of intracellular yeast and significantly reduced nitric oxide production. These results show that IFN-γ-activated murine Mφs killP. brasiliensisconidia through thel-arginine–nitric oxide pathway.